体外成熟液中添加PAF对牦牛卵母细胞发育能力及基因表达的影响

doi:10.11843/j.issn.0366-6964.2020.03.011

摘要/Abstract

摘要： 旨在探讨体外成熟（IVM）液中添加血小板活化因子（PAF）对牦牛卵母细胞发育能力及基因表达影响。本试验将牦牛1 025个卵丘-卵母细胞复合体（COCs）分为4组，分别在含不同浓度PAF（0、10^-8、10^-7和10^-6mol·L^-1）的IVM液中成熟后，与奶牛精子体外受精（IVF）和体外培养（IVC），比较分析各组卵母细胞成熟率、卵裂率和囊胚率以及COCs和胚胎中抗凋亡（BCL-2）、促凋亡（BAX）、表皮生长因子（EGF）及其受体（EGFR）、转录因子（OCT-4和NANOG）和c-fos等基因的表达量差异。结果表明，IVM液中PAF浓度为10^-7mol·L^-1组的卵母细胞成熟率（（92.16±0.19）%）、卵裂率（（77.20±0.85）%）和囊胚率（（46.71±0.68）%）显著高于其他组（P<0.05）。qPCR结果显示，在该组成熟的COCs中BCL-2、EGF、EGFR、c-fos表达量显著上调（P<0.05），而促凋亡基因BAX表达量显著下调（P<0.05），囊胚中EGF、EGFR、OCT-4基因表达量显著上调（P<0.05）。各组间2-、4-、8-细胞、桑椹胚和囊胚中BCL-2、BAX和NANOG表达量差异不显著，但桑椹胚和囊胚中BCL-2表达量均明显下调，而BAX表达量均明显上调。综上所述，IVM液中适宜浓度PAF（10^-7mol·L^-1）可以促进牦牛卵母细胞成熟和胚胎发育，调控细胞凋亡和增殖相关基因的表达。

关键词: 牦牛, 血小板活化因子, 卵母细胞, 体外受精, qPCR

Abstract: The objectives of this study were to evaluate the effects of platelet-activating factor (PAF) supplementation in in vitro maturation (IVM) medium on yak (Bos grunniens) oocyte development competence and gene expression. A total of 1 025 yak cumulus-oocyte complexes (COCs) were assigned to 4 groups. COCs in different groups were matured in IVM medium with different concentrations of PAF (0, 10^-8, 10^-7, and 10^-6mol·L^-1), followed by in vitro fertilization (IVF) with cattle sperm and in vitro culture (IVC). The maturation rate, cleavage rate and blastocyst rate of oocytes, and the relative expression level of pro-apoptosis (BAX), anti-apoptosis (BCL-2), epidermal growth factor (EGF) and its receptor (EGFR), transcription factors (OCT-4 and NANOG) and c-fos genes in COCs and embryos were compared among different groups. The results showed that oocytes in IVM medium with 10^-7mol·L^-1 PAF had higher maturation rate((92.16±0.19)%), cleavage rate((77.20±0.85)%) and blastocyst rate((46.71±0.68)%) than those in the other groups (P<0.05). The results of qPCR showed that the expression level of BCL-2, EGF, EGFR, c-fos were significantly up-regulated (P<0.05), but the expression level of pro-apoptotic gene (BAX) was significantly down-regulated in matured COCs (P<0.05) in 10^-7 mol·L^-1 PAF group. The expression level of EGF, EGFR and OCT-4 in blastocysts were significantly up-regulated in 10^-7 mol·L^-1 PAF group (P<0.05). The expression level of BCL-2, BAX and NANOG genes in 2-, 4-, 8-cell, morula and blastocyst stages were not different among different groups, but the expression of BCL-2 was down-regulated and BAX was up-regulated in morula and blastocyst. In conclusion, the optimal PAF concentration in IVM medium (10^-7mol·L^-1) could improve oocyte maturation and embryo development, and regulate the expression of apoptosis and proliferation related genes of the yak.

Key words: yak, platelet-activating factor, oocyte, in vitro fertilization, qPCR

中图分类号:

S823.85

王琴, 熊燕, 字向东. 体外成熟液中添加PAF对牦牛卵母细胞发育能力及基因表达的影响[J]. 畜牧兽医学报, 2020, 51(3): 514-523.

WANG Qin, XIONG Yan, ZI Xiangdong. Effects of PAF Supplementation during in vitro Maturation Medium on Yak Oocyte Development Competence and Gene Expression[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 514-523.

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